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Hepatology. 2011 Oct;54(4):1351-9. doi: 10.1002/hep.24490. Epub 2011 Aug 9.

Efficient production of Fah-null heterozygote pigs by chimeric adeno-associated virus-mediated gene knockout and somatic cell nuclear transfer.

Author information

1
Papé Family Pediatric Research Institute & Oregon Stem Cell Center, Oregon Health & Sciences University, Portland, OR 97239, USA. hickeyr@ohsu.edu

Abstract

Hereditary tyrosinemia type I (HT1) results in hepatic failure, cirrhosis, and hepatocellular carcinoma (HCC) early in childhood and is caused by a deficiency in the enzyme fumarylacetoacetate hydrolase (FAH). In a novel approach we used the chimeric adeno-associated virus DJ serotype (AAV-DJ) and homologous recombination to target and disrupt the porcine Fah gene. AAV-DJ is an artificial chimeric AAV vector containing hybrid capsid sequences from three naturally occurring serotypes (AAV2, 8, and 9). The AAV-DJ vector was used to deliver the knockout construct to fetal pig fibroblasts with an average knockout targeting frequency of 5.4%. Targeted Fah-null heterozygote fibroblasts were used as nuclear donors for somatic cell nuclear transfer (SCNT) to porcine oocytes and multiple viable Fah-null heterozygote pigs were generated. Fah-null heterozygotes were phenotypically normal, but had decreased Fah transcriptional and enzymatic activity compared to wildtype animals.

CONCLUSION:

This study is the first to use a recombinant chimeric AAV vector to knockout a gene in porcine fibroblasts for the purpose of SCNT. In using the AAV-DJ vector we observed targeting frequencies that were higher than previously reported with other naturally occurring serotypes. We expect that the subsequent generation of FAH-null homozygote pigs will serve as a significant advancement for translational research in the areas of metabolic liver disease, cirrhosis, and HCC.

PMID:
21674562
PMCID:
PMC3184202
DOI:
10.1002/hep.24490
[Indexed for MEDLINE]
Free PMC Article

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